The present invention relates to a method for preventing or activating the migration of cells in a mammal, in particular a human. The method comprises the use of synthetic peptides, or of defined recombinant soluble forms of the urokinase receptor to activate the recruitment of inflammatory cells in a mammal, by stimulating a cellular adaptor that mediates the chemotactic activity of uPA. This mechanism does not require the protease activity of urokinase. Actually it by-passes it by acting on a downstream step, namely the interaction of such a peptide or recombinant soluble form of urokinase receptor with a cellular adaptor. Additionally, the reagents produced are also useful to identify and isolate drugs that can inhibit said processes of cell recruitment, and hence may be employed in blocking the malignant phenotype of cancer and of aggressive hyper-or auto-inflammatory diseases.
Cell Migration and Disease
The response of the human body to noxic stimuli is dependent on the local production of inflammatory molecules as well as on the recruitment of specialized cells that migrate from the blood and the neighbouring tissue into the damaged site. These cells, neutrophils, monocyte-macrophages, lymphocytes and endothelial cells, respond to specific migratory stimuli and build up a defense response that results in the destruction and elimination of the nocive agent or organism. While essential and beneficial for the host defense, this type of response may also prove dangerous for the host or may hamper the success of some therapeutic or preventive approaches like transplantation and vaccination. In fact, a series of severe pathological entities exist which can be ascribed to excess or deficient migratory cell response. For example, immunodeficient patients fail to respond to infective agents and this may be due to inability of the cells to rush to the damaged or infected site. Excess of recruitment, on the other hand, may be responsive of destructive pathologies in which defense cells attack cells and functions of the host, as it happens for example in autoimmune diseases. Failure to properly respond to vaccination, moreover, may also depend on unwanted recruitment of host inflammatory cells that destroy the antigenic cells too quickly to promote an immunological response. The availability of a specific xe2x80x9cadjuvantxe2x80x9d to natural immunity cells, would be advantageous as it would produce a stronger antibody response.
The malignancy of cancer cells mostly consists in the ability to invade and metastasize at a distance; in this process, however, non-malignant stromal host cells actively participate in establishing the malignant phenotype and hence the destructive and invasive phenotype. While the mechanisms involved are largely unknown, it is clear that stromal cells not only respond to stimuli arriving from the cancer cells but also signal to cancer cells through mechanisms of their own. In order to block the invasive phenotype of cancer, it is thus essential to act both on cancer and on stromal cells.
The Urokinase/urokinase Receptor System
The activity of urokinase (uPA) can be confined to the cell surface by the presence of a specific receptor (uPAR, CD87). uPA is a serine protease important in maintaining the fibrinolytic state of the body as it generates plasmin from plasminogen and hence prevents fibrin deposition. Plasmin is a broad spectrum protease that can destroy many proteins of the extracellular matrix and hence the inter-cellular and cell-to-extracellular matrix connections. UPA and uPAR have long been recognised as regulators of cell migration and hence to be important in inflammation and cancer invasion. In addition to functions connected to its proteolytic activity, uPA has other properties that do not require proteolytic activity, but simply the binding to the receptor. In fact, the proteolytic and the receptor binding activities are separated on the uPA molecule and can be assayed individually (receptor binding in the amino terminal fragment, proteolysis in the carboxy-terminal fragment). Among the functions that do not require the proteolytic moiety of uPA, are the stimulation of mitogenesis, cell migration, adhesion and, in particular, chemotaxis (Gudewicz and Bilboa, 1987; Gyetko et al., 1994; Resnati et al., 1996; Besser et al., 1996).
The specific uPA receptor (uPAR) is a GPI-anchored plasma membrane protein endowed with a very high affinity (Kd of 0.1-1 nM) for uPA, pro-uPA and inhibited forms of uPA, like the uPA-PAI-1 complex (Fazioli and Blasi, 1994). In addition to uPA, uPAR also binds vitronectin with an about 10-20 nM affinity as well as integrins. Structurally, uPAR is formed by three repeats of about 90 amino acid residues connected by two linker regions (Danxc3x8, Blasi et al., 1990; Behrendt et al., 1991) which define functionally and structurally different domains: the amino terminal domain (D1) contains the uPA binding site, while the carboxyterminal region containing domains D2 and D3 binds vitronectin. However, the integrity of the three-domains structure is necessary for high affinity binding to uPA at least with the purified, soluble protein (Danxc3x8 et al., 1994). To date, no information has been provided as to the function of the linker regions.
uPAR is expressed in circulating blood cells, in particular monocytes, neutrophils and T-lymphocytes but not in erythrocytes or B-lymphocytes., In addition, uPAR is a target gene in lymphocytes and macrophage activation (CD87). Indeed, monocytes and monocyte-like cells (like HL60, U937) express uPAR, or are induced to overexpress uPAR by a variety of cytokines and other agents, like phorbol ester PMA, phytohemagglutinin, bacterial liposaccharide, TGFb1/vitamin D3, GM-CSF, IFNg, TNFa and others. In human T-lymphocytes, stimulation of the TCR/CD3 complex, lymphokines IL2, IL4, IL7 or the concomitant activation of the T cell receptor and integrins engagement, all induce uPAR expression (Nykjmr et al., 1994; Bianchi et al., 1996). It is also noteworthy, that tumor infiltrating T-lymphocytes heavily express UPA-R and some of the properties of the activated T-lymphocytes, in particular their migration through reconstituted basement membranes, appear to be at least in part uPA- and uPAR-dependent (Bianchi et al., 1996). In view of the important contribution of stromal cells to the invasiveness of cancer, it is also important to stress that macrophages present in human breast cancer and other cancers, where the level of uPAR production by the overall tumor importantly contributes to its malignancy, express high levels of uPAR (Brunner et al., 1996). The cooperation between stromal and cancer cells in cancer invasiveness, implies that high expression of uPAR by cancer or stromal cells, can affect cell prognosis possibly through different mechanisms depending on the overexpressing cell-type.
uPA/uPAR and Chemotaxis
Cooperation between cancer and stromal cells poses the problem of how uPAR expression on stromal cells influences the malignancy of cancer cells. Since the uPA/uPAR system has chemotactic activity, the production of uPAR by stromal cells can attract cancer cells through a chemokine like action. Indeed, chemokines produced by certain cells are anchored to a presentation molecule in the extracellular matrix and attract other cells having specific receptors (Schall and Bacon, 1996). The information available on the uPA/uPAR system supports this possibility.
uPA/uPAR and Chemotaxis
Stimulation of chemotaxis in monocyte-like cells, fibroblasts and some cancer cells requires the specific cell surface uPAR (CD87) which mediates the chemoattractant activity of uPA (Resnati et al., 199.6). Chemotaxis by uPA does not require its protease activity, but only the occupancy of its receptor, and can be reproduced with the enzymatically inactive receptor binding moiety ATF (amino terminal fragment), pro-uPA and by chymotrypsin-cleaved soluble uPAR (Resnati et al., 1996). In mouse, the importance of this system in cell recruitment in vivo is shown by the fact that uPA is essential for the inflammatory response; indeed, mice lacking the uPA gene are highly defective in the response to bacterial and possibly other infections, and are incapable of recruiting T-lymphocytes and macrophages to the site of infection (Gyetko et al., 1996). In man, uPA and uPAR are induced during T cells activation and directly contribute to the in vitro migration of human T cells (Nykjxc3xa6r et al., 15 1994; Bianchi et al.,1996). Moreover, tumor infiltrating T-lymphocytes heavily express uPAR (Bianchi et al., 1996).
uPA-dependent chemotaxis is mediated by the activation of src-family tyrosine kinases (i.e. Hck in monocytes and Src in fibroblasts). The mechanism involved requires that uPA modifies uPAR conformation such that it can bind to a still unidentified adaptor. Indeed, in cells lacking uPAR, a mixture of fragments generated by cleaving a soluble, recombinant uPAR with chymotrypsin, has a very potent chemoattractant activity (IC50 of 10-20 pM) (Resnati et al., 1996).
In Boyden chamber-based assays, uPA or its derivatives induce migration through a chemotactic gradient in bovine adrenal capillary endothelial cells, keratinocytic cell lines, monocytes, monocyte-like cells, fibroblasts cell lines, and in neutrophils both in vitro and in vivo (Besser et al., 1996; Resnati et al., 1996). The involvement of uPAR in chemotaxis is not only a direct one, but may also be indirect by interfering with the chemotaxis induced by other chemokines. Exposure of human neutrophils to a chemotactic gradient of FMLP or MCP-1 chemokine localizes uPAR to the leading edge of the migrating cell; anti-uPAR antibodies ablate the chemotactic activity. The chemotactic effect of FMLP or MCP-1 does not require the proteolytic activity of uPA, but uPAR occupancy is absolutely required for chemotaxis as uPAR antibodies or antisense RNA expression totally abolishes FMLP or MCP-1 activity.
The steps between the binding of chemokines to chemokine receptors and cell movements, are many and complex: signal transduction, reorganization of cytoskeleton, formation of focal adhesions, attachment and detachment from the substrate with pseudopodal extension and retraction to effect directional migration (Premack and Schall, 1996). The chemotactic activity of uPAR has many of the properties of chemokines. Interaction between uPAR and integrins has been observed in neutrophils: the leucocyte integrin CD11b/CD18, aMb2, also known as the complement receptor type-3 (CR3) has been shown to physically associate with uPAR. This interaction is reversible and correlates with cell shape. In resting cells, uPAR and CR3 are co-localized, but following spontaneous cell polarization and migration, the two receptors dissociate, CR3 concentrates in the uropods and uPAR in the lamellipodia of polarized cells. Since CR3 regulates cell adhesion, chemotaxis and cell migration into inflammatory sites, the interaction with uPAR may be central to all these functions and hence to the cell-recruitment action of uPA/uPAR. No information however is available as to whether this integrin (or others) is directly involved in mediating the uPAR chemotactic signal, i.e. whether this integrin is the so far unidentified adaptor.
Activation of migration by uPAR occupancy is observed also in epithelial cell lines where uPAR associates with a protein kinase that can serine-phosphorylate two cytokeratins, CK18 and CK8. The enzyme responsible is possibly the protein kinase Cz, since the process is independent from Ca2+, resistant to PMA down modulation and since the kinase can be specifically immuno-recognized. In vivo, CK18 and CK8 are phosphorylated on serine and uPAR occupancy elicits a time-dependent increase in phosphorylation of CK8, cell shape changes and redistribution of cytokeratin filaments. Pro-uPA treatment caused rounding up of the cells and reduction of their diameter. In control cells, cytokeratins were distributed in a lattice array parallel to the surface of the coverslips; upon treatment with pro-uPA, little or no cytokeratins were observed in filament form. These data tie together the migratory effect of pro-uPA, the activation of protein kinase C and the cell shape changes. Tyrosine-phosphorylation of a non-characterized 35 kDa protein occurs upon binding of pro-uPA to U937 myeloid cells. In these cells, occupancy of uPAR promotes cell adhesion during PMA-induced differentiation. It has been shown that uPAR directly binds vitronectin and promotes cell adhesion. Moreover, uPAR directly interferes with the substrate recognition of adhesion receptors by forming complexes directly with the beta integrins, and hence promoting adhesion on vitronectin and inhibiting adhesion on fibronectin (Wei et al., 1996). Indeed, uPAR can be co-immunoprecipitated with anti-integrins IgG. The effect of uPAR on cell adhesion is also in line with its chemokine-like activity. A direct interaction of uPAR with integrins is suggested by their co-immunoprecipitation and by the finding of peptides that specifically inhibit the co-immunoprecipitation without interfering with the binding of uPA or vitronectin to uPAR (Wei et al., 1996). Therefore, integrins might represent the described adaptor suggested by Resnati et al. (1996). In this respect, the uPAR-like small molecules to be described in this application, have whatsoever no connection, either structural or conceptual, with the peptide identified by Wei et al. (1996).
In monocytes and fibroblasts, occupancy of uPAR induces chemotaxis and the effect does not require extracellular proteolysis (Resnati et al., 1996). In this system, ATF binding causes a time-dependent and transient activation of a tyrosine kinase of the Src family, the p56/p58 Hck. In addition, uPAR itself associates with tyrosine kinases, through what appears to be a transmembrane-adaptor-mediated mechanism. Indeed, cells lacking uPAR, and therefore not responsive to ATF, are capable of chemotaxis when challenged with a mixture of chymotryptic fragments of soluble uPAR (chymotrypsin cleaves uPAR in two fragments between domain D1 and D2) (Resnati et al., 1996). This effect also occurs in fibroblasts derived from uPARxe2x88x92/xe2x88x92mice. Therefore, uPAR can recognize a cell surface molecule which in turn is capable of mediating signal transduction. The occupancy of surface uPAR must in fact expose binding sites for the unidentified adaptor and hence transform uPAR from a receptor into a ligand. The role of tyrosine kinases in uPAR-dependent chemotaxis is also shown by the effect of tyrosine kinase inhibitors that prevent the chemotactic response to ATF, and by the failure of primary srcxe2x88x92/xe2x88x92fibroblasts to respond to the soluble, cleaved uPAR (Resnati, Blasi and Fazioli, unpublished). Interestingly, while uPA binding to uPAR is strictly species-specific, human uPAR acts as a chemoattractant as efficiently on human as on murine cells. In particular, chymotrypsin-cleaved soluble uPAR has an IC50 of about 20 pM (Resnati et al., 1996).
The Chemoattractant Property of uPAR as a Target for Novel Drugs Acting Downstream of the uPA/uPAR Interaction
The uPA/uPAR system is involved in cancer cell invasiveness, and also in inflammation and other diseases (Danxc3x8 et al., 1990). And in fact, the block of uPA/uPAR interaction by specific antagonists, or the reduction of uPAR synthesis by antisense gene expression leads to a drastic reduction of the migratory properties of cancer cells (Fazioli and Blasi, 1994). In human cancer, the expression level of the components of the system, uPA, PAI-1 and uPAR, directly relates to the malignancy of the disease and its prognosis (Brxc3xcnner et al., 1996). In fact, many if not all solid cancers have already released metastatic cells which are found in the bone marrow. The presence of uPAR-positive metastatic cells at the time of diagnosis represents a sign of extreme likelihood of poor prognosis, while the presence of metastatic cells per se does not.
On the basis of the above, uPAR appears to be a specific target for novel types of drugs that might interfere with the process of inflammation or of cancer invasiveness. Two different possibilities are open: to block uPAR by interfering with uPA binding (uPAR antagonists), or to prevent the direct transduction of a migratory signal, via interaction with other molecules, bypassing the step of the interaction between uPA and uPAR. Drugs preventing the binding of uPAR to the before mentioned adaptor should also prevent chemotaxis via uPAR. Therefore, antagonists of the uPAR/adaptor interaction should block or decrease the migration of cells and hence might be useful in diseases where this decrease or block is advantageous, like cancer, autoimmune and excess-inflammatory reactions.
uPAR as Target for Drugs Enhancing the Recruitment of Inflammatory Cells
The recruitment of cells via the uPA-uPAR interaction suggests that drugs should be found which promote cell recruitment by binding to uPAR. And indeed, uPA derivatives like the amino terminal fragment ATF, pro-uPA or possibly also synthetic peptides that cover the uPAR-binding region of uPA are in fact capable of substituting for uPA in various functions not requiring uPA proteolytic activity (see for example, Fazioli and Blasi, 1994; Besser et al., 1996, Resnati et al., 1996). On the other hand, since uPA binding to uPAR causes a conformational change that transforms uPAR from a receptor for uPA into a ligand for a still unidentified membrane adaptor (Resnati, et al. 1996), an alternative possibility of enhancing cell recruitment would be to bypass the first step, i.e. the uPA-uPAR binding step, and stimulate cell migration by acting at the level of the unidentified adaptor with uPAR itself or specific uPAR-like agonists. These agonists might therefore be useful in diseases in which the recruitment of cells is blocked by genetic or acquired malfunctions upstream of the uPAR/adaptor interaction. Also, the availability of such drugs might be advantageous in connection with increasing the immunogenicity of various antigens, for example in vaccination, as it might act like an adjuvant by recruiting cells responsible of natural immunity.
uPAR as Target for Controlling HIV Infection
uPAR is an activation antigen in both T-lymphocytes and in monocytes (Nykjar et al., 1994; Bianchi et al., 1996), i.e. in cells capable of active migration and in which HIV can replicate. While CD3-positive circulating lymphocytes do not or only weakly express uPAR, CD8+ T lymphocytes of patients of AIDS and other viral diseases display a high expression of uPAR, and in several of these patients as many as 80% of their T-lymphocytes are high expressers (Nykjar et al., 1994). A fraction of persons remain uninfected by HIV despite multiple high-risk sexual exposure, and this is connected with the presence of HIV suppressive facfors in their blood (Paxton et al., 1996). Several chemokines have been identified as HIV-suppressive facfors produced by CD8+ T cells (Cocchi et al., 1996) and these chemokines represent novel targets for AIDS therapy. Soluble forms of uPAR exist in the blood and uPAR is in fact cleaved in cell lines and in cancer tissues. Since cleavage activates the chemotactic activity of soluble uPAR (Resnati et al., 1996), these soluble, cleaved forms might, like actual chemokines, also interfere with the infectivity or survival of HIV. If a cleaved form of soluble uPAR is also involved in HIV infectivity or resistance, it might represent a target for a novel type of therapy in cases of AIDS.
uPAR as Target for Drugs Enhancing Wound Healing
Excessive fibrin deposits are observed in plasminogen-deficient, uPA-deficient and in the double uPAR/tPA-deficient mice and these mice are also deficient in skin wound healing (Carmeliet and Collen, 1996). Several mechanisms may be active in this phenomenon, including chemotaxis, since uPAR is localized at the leading edge of migrating keratinocytes during re-epithelization of mouse skin wounds. The importance of monocytes and macrophages in the process of wound healing makes it even more likely that this process might be augmented by a uPAR agonist capable of reproducing its chemoattractant property.
Novel Approach to Blocking uPAR
So far, approaches directed to block the effect of uPAR in cell migration have been limited to the search of uPAR antagonists, i.e. drugs that prevent the binding of uPA to uPAR. In general, such drugs are also expected to inhibit cell surface proteolytic activity. However, binding of uPA to uPAR stimulates chemotaxis by transforming this molecule from a receptor into a ligand for an adaptor (Resnati et al., 1996). Therefore, the identification of a small molecule that can mimic the chemotactic activity of uPAR gives a new handle to control the uPAR-dependent reactions, independent of its occupancy by uPA. In relation to the present invention, such small molecules have been discovered and characterized. They act downstream of the uPA/uPAR interaction and therefore have no effect on cell surface proteolytic activity. These small molecules can be applied both in cases of deficient cell recruitment and migration (as stimulators of the process), and in cases of excess cell recruitment and migration. In the latter case, the uPAR-like small molecules can be employed to identify suitable inhibitors of the uPAR-adaptor interaction.
uPA is essential for inflammatory cell recruitment, binds uPAR and induces chemotaxis by direct signalling in a uPAR-dependent way. In this function uPAR interacts with an unidentified adaptor molecule, and in fact a mixture of chymotryptic fragments of soluble uPAR has chemoattracting activity. The present invention is based upon the discovery of the mechanism through which uPAR activates chemotaxis. First the region of uPAR inducing chemotaxis was identified. As shown in example 1, soluble fragments of uPAR were produced either by separation of chymotrypsin-cleaved uPAR, or by recombinant DNA technology, and were used in a chemotaxis assay to locate the region of uPAR responsible for the chemoattracting activity on THP-1 cells. Such a region (residues 88-92 of uPAR) was localized in the linker region between domain D1 and D2. In fact, the chemotactic activity was found either in domain D1 or D2, whenever they included this linker region: carboxyterminally of domain D1, or aminoterminally of domain D2. In example 2, the chemotactic activity of uPAR was reproduced using synthetic peptides including the 88-92 uPAR sequence of uPAR, on human THP-1 monocytoid cells (and murine LB6 fibroblastic cells). Synthetic molecules (peptides 1 and 2) including the sequence SRSRY (ser-arg-ser-arg-tyr) (SEQ ID NO:7) had a very potent chemotactic activity at concentrations as low as 0.1 pM. Control peptides had no activity, in particular a peptide covering the carboxyterminal sequence deduced from uPAR cDNA, and a peptide having the same amino acid composition as peptide 1 but a scrambled sequence. In example 3, we show that peptide 1 stimulates chemotaxis through the same mechanism employed by the ligand uPA, or by the mixture of chymotrypsin fragments of the soluble uPAR. Peptide 1, in fact, activates Hck tyrosine kinase of THP-1 cells with the same time-course and concentration dependence observed with the mixture of chymotrypsin fragments of soluble uPAR. Example 4 shows how to practically identify inhibitors of uPAR by employing a simple panning assay using uPAR mimicking peptides, i.e. peptide 1 and 2 of the invention.
One aspect of the present invention relates to a method of stimulating or increasing the chemotactic activity of a cell, comprising adding a chymotrypsin-cleaved suPAR peptide or a functional analogue of said peptide.
Within the scope of the uPAR peptides according to the present invention are uPAR peptides derived from uPAR found in mammalian species, such as human, bovine, murine and rat.
In the present context, the term xe2x80x9cfunctional analoguexe2x80x9d means a peptide with an amino acid sequence which is not identical to a chymotrypsin-cleaved suPAR peptide, but which has a substantially identical effect with respect to stimulating or increasing the chemotactic activity of a cell. This effect will typically be tested in an assay such as the one described in example 1. The increase in chemotactic activity in such an assay is preferably at least 100% of control, more preferably at least 200% of control, even more preferably at least 400% of control.
Non-limiting examples of functional analogues of the human uPAR chemotatactic activity are:
ser-arg-ser-arg-tyr (SEQ ID NO:7)
ser-arg-asn-arg-tyr (SEQ ID NO:8)
ser-arg-gly-arg-tyr (SEQ ID NO:9)
ser-gln-ser-arg-tyr (SEQ ID NO:10)
ser-gln-asn-arg-tyr (SEQ ID NO:11)
ser-gln-gly-arg-tyr (SEQ ID NO:12)
pro-arg-ser-arg-tyr (SEQ ID NO:13)
pro-arg-asn-arg-tyr (SEQ ID NO:14)
pro-arg-gly-arg-tyr (SEQ ID NO:15)
pro-gln-ser-arg-tyr (SEQ ID NO:16)
pro-gln-asn-arg-tyr (SEQ ID NO:17)
pro-gln-gly-arg-tyr (SEQ ID NO:18)
Other molecules which contain the above listed functional analogues and have an effect in a chemotactic assay will also be functional analogues within the scope of the present invention, as well as peptides or peptoids having a similar sequence (e.g. 400%-80% sequence identity, such as 60%, 66%, 75% or 80% sequence similarity with any of the listed peptides) as any of the listed peptides and which exhibit an effect in the assay.
Another aspect of the present invention thus relates to a peptoid analogue of the peptide with the sequence SRSRY (SEQ ID NO:7) comprising one or more non-naturally occurring amino acids. The peptoid analogues according to the invention can for example be prepared by using peptide synthesis methods, such as the method described by Merrifield. 1963. The synthesis may include any number of non-naturally occurring amino acids and optionally also including one or more naturally occurring amino acids in the preparation. The peptides and functional analogues of the invention can be added, in an effective amount, directly to cells for to an individual, such as a human patient, who harbours the cells in which chemotactic activity can be stimulated or increased.
A very important aspect of the present invention relates to peptides of the invention. Such peptides are a peptide with the sequence SRSRY (SEQ ID NO:7) or a functional analogue thereof and a peptide with the sequence AVTYSRSRYLEC (SEQ ID NO:1) and a peptide with the sequence SRSRYLEC (SEQ ID NO:3).
A particularly preferred embodiment of the present invention relates to a recombinant fusion protein, comprising one or more of the peptide species according to the invention and optionally additionally comprising one or more of the peptide species according to the invention.
Functional analogues, according to the invention, which are prepared using peptide synthesis are within the scope of the present invention. Functional analogues, according to the invention, which are prepared by using other methods than peptide synthesis, such as recombinant expression, are also within the scope of the present invention.
A further aspect of the present invention is a method of stimulating or increasing a local inflammatory response, comprising adding a chymotrypsin-cleaved fragment, or mixture of fragments, of uPAR, said fragment or mixture of fragments comprising an SRSRY (SEQ ID NO:7) peptide, or a functional analogue of said peptide.
In the present context, the term xe2x80x9cstimulating or increasing local inflammatory responsexe2x80x9d means that one or more characteristics of an inflammatory response can be measured to be stimulated or increased. Such characteristics will typically be release of cytokines by T-lymphocytes or monocyte-macrophages, increase of chemotactic activity, ability to cause a local recruitment of lymphocytes/monocytes upon subcutaneous administration in a suitable laboratory animal, etc. In this particular case, the use of uPA-deficient mice is particularly favourable as these mice have a very low anti-inflammatory response. Methods to measure these activities are available routinely, and increases of such activities as mentioned of at least 50% are considered significant, preferably at least 80% even more preferably at least 100%.
Another important aspect of the present invention is a method of stimulating or increasing the chemotactic activity of a cell, comprising adding a peptide comprising the sequence SRSRY (SEQ ID NO:7)or a functional analogue of said peptide.
A further aspect of the present invention is a method of stimulating or increasing wound healing, comprising adding a chymotrypsin-cleaved suPAR peptide or a functional analogue of said peptide. A peptide comprising the sequence SRSRY (SEQ ID NO:7) or a functional analogue of said peptide can be added according to the invention.
In the present context, the term xe2x80x9cstimulating or increasing wound healingxe2x80x9d means that at least one of the physiological characteristics or processes involved in healing of various types of wounds in an individual, such as a human, is substantially stimulated or increased. One way of quantifying such stimulation or increase in wound healing comprises measuring wound healing in a plasminogen-deficient or even a normal mouse, and identifying the time required for 50% healing. A reduction of the time of healing, upon addition of an effective amount of a composition comprising suPAR peptide, of at least 20%, preferably at least 50%, would represent a substantial wound healing stimulation.
An important aspect of the present invention relates to a method of stimulating the kinase activity of p56/p59hck comprising adding a chymotrypsin-cleaved suPAR peptide or a functional analogue of said peptide or comprising adding a peptide comprising the sequence SRSRY (SEQ ID NO:7) or a functional analogue of said peptide.
In the present context, the term xe2x80x9cstimulating the kinase activityxe2x80x9d means substantially increasing the ability of p56/p59hck to phosphorylate a protein substrate. This will typically be measured using in vitro assays as the ones described in example 3. Quantification of such an effect might be obtained by cutting the bands from a gel such as the one showed in FIG. 5, and subsequently measuring the 32P-radioactivity, after normalization.
A very important aspect of the present invention relates to a method of testing whether a compound is capable of stimulating the binding between a chymotrypsin-cleaved soluble form of u-PAR and a cellular adaptor, said method comprising adding an effective amount of the compound to a test system comprising cells lacking endogenous u-PAR and measuring chemotactic activity; if chemotactic activity is present, then the compound is capable of stimulating said binding.
In the present context the term xe2x80x9cstimulating the bindingxe2x80x9d should be taken to mean increasing the binding in an assay, substantially resembling the above described, by at least 20%, preferably at least 30%, more preferably by at least 40%, even more preferably by at least 60%.
In another aspect, the present invention relates to a compound which is capable of stimulating the binding between a chymotrypsin-cleaved soluble form of uPAR and a cellular adaptor which has been selected by the method of the invention as described above.
A particularly important aspect of the present invention relates to a method of testing whether a compound is capable of blocking or inhibiting the binding between a chymotrypsin-cleaved soluble form of uPAR and a cellular adaptor, said method comprising adding chymotrypsin-cleaved soluble form of uPAR and an effective amount of the compound to a test system comprising cells lacking endogenous uPAR and measuring chemotactic activity. If chemotactic activity is inhibited or prevented, then the compound is capable of blocking said binding.
In the present context the term xe2x80x9cblocking or inhibiting the bindingxe2x80x9d should be taken to mean decreasing the binding in an assay, substantially resembling the above described, by at least 20%, preferably at least 30%, more preferably by at least 40%, even more preferably by at least 60%.
In a further aspect, the present invention relates to a compound which is capable of blocking or inhibiting the binding between a chymotrypsin-cleaved soluble form of uPAR and a cellular adaptor which has been selected by the method of the invention as described above.
An important embodiment of the present invention is a compound according to the invention for the treatment of cancer, autoimmune disease and/or hyper-inflammatory diseases. A compound which is capable of blocking or inhibiting the binding between a chymotrypsin-cleaved soluble form of uPAR and a cellular adaptor would according to the invention be administered to an individual, such as a human patient, in need thereof in a pharmaceutically acceptable form and in a pharmaceutically effective amount.
A very important aspect of the present invention relates to a method of screening compounds for potential anti-inflammatory or anti-migratory properties. The method according to the invention comprises
(a) coating a plastic surface which does not normally bind mammalian cells with an effective amount of a peptide according to the invention,
(b) exposing selected mammalian cells to the coated surface for 30-300 minutes at 37 C., in the presence or absence of a potentially inhibitory compound, and
(c) testing the ability of the cells to bind to the peptide coated surface by microscopic inspection or Coomassie Blue staining of the plates; if cellular binding is inhibited or prevented, then the compound is capable of blocking said binding and is a potential anti-inflammatory or anti-migratory agent.
In a further aspect, the present invention relates to oligo nucleotides, in particular oligonucleotides encoding the peptides given by SEQ ID NO: 1 and SEQ ID NO: 3.
Another aspect of the present invention relates to the use of an oligonucleotide according to the invention, for the recombinant expression of peptides for use in the screening of compounds according to the invention.
Preferred embodiments of the present invention relate to use of oligonucleotides according to the invention, wherein the recombinant expression is performed in an expression system chosen from the group consisting of an E. coli expression system, a yeast expression system, a mammalian expression system or an insect cell expression system.
When using the oligonucleotides according to the invention as the basis for preparing peptides according to the invention by performing recombinant expression, it is preferred to combine said oligo nucleotides with nucleic acid sequences, such as regulatory sequences, which enable. the expression system of choice to express large amounts of peptide.
A particularly important aspect of the present invention relates to a method of stimulating or increasing anti-tumour immunity in autologous bone marrow transplantation treatment of an individual, such as a human patient, bearing tumours, comprising modifying tumour cells by transfecting them with a eukaryotic vector expressing a nucleic acid sequence encoding the peptide SRSRY (SEQ ID NO:7), or a functional analogue thereof.
In the present context, the term xe2x80x9canti-tumor immunityxe2x80x9d means the set of cellular and chemical reactions which an organism can organize with the aim of rejecting a tumor. In this respect, the production of cytokines and chemokines is of major importance as it will drive the recruitment and the functional activation of cells that can specifically attack the tumor cells and hence destroy it.
Another important aspect of the present invention relates to a method of stimulating or increasing cellular immunity in an individual, such as a human patient, who is immunodeficient, said method comprising adding a chymotrypsin-cleaved suPAR, said suPAR comprising the peptide SRSRY (SEQ ID NO:7), or a functional analogue thereof.
In the present context, the term xe2x80x9ccellular immunityxe2x80x9d means the recruitment and the functional activation of immune cells, such as T-lymphocytes, NK-cells, B-lymphocytes, macrophages etc.
In the present context, the term xe2x80x9cimmunodeficientxe2x80x9d means that the immune system of an individual is compromised and thus can be said to function at a sub-normal level with respect to at least some of the systems reactions.
Yet another aspect of the present invention relates to a method of vaccinating a subject, such as a human patient, suffering from tumours, with the aim of stimulating an anti-tumour T cell response, said method comprising vaccination of said subject with a human tumour cell line compatible on both the histotype and the HLA-A2 basis and modified so as to release the chemotactic region of the human or murine or bovine or rat uPAR, said chemotactic region being a fusion peptide or a domain of uPAR containing the SRSRY (SEQ ID NO:7) peptide or a functional analogue thereof.
A preferred embodiment of the present invention relates to a method according to invention, wherein vaccination is carried out by using autologous tumor cells.
A very important aspect of the present invention relates to a method of exploiting the cellular immunity of uPAR derivatives or peptides of the present invention to kill tumour cells, said method comprising injecting a tumour-bearing subject, such as a human patient, with tumour-targeted cells modified to express the chemotactic region of uPAR from human, bovine, murine or rat, or any other uPAR having substantially the same properties, said method further comprising fusing the target vector to said region, being a peptide or a domain of uPAR containing the SRSRY (SEQ ID NO:7) peptide, or a functional analogue thereof.
In the present context the term xe2x80x9cuPAR derivativesxe2x80x9d is used as defined in WO 90/12091, whereas the term xe2x80x9ctumour-targeted cellsxe2x80x9d means cells that, naturally or because they are specially engineered, can be injected into an individual and will specifically interact with tumour cells of said individual.